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The effect of cell shape, cell density, contact with extracellular matrix and cell polarity on the phenobarbital (PB)-induced gene expression of CYP2B1 and CYP2B2 (CYP2B1/2B2) in adult rat hepatocytes was investigated. High cell density enhanced the induction of CYP2B1/2B2 gene expression by PB. Hepatocytes cultured on EHS gel showed a spherical cell shape and highly enhanced the induction of CYP2B1/2B2 gene expression by PB. Although monolayer hepatocytes cultured on type I collagen (TIC) and type IV collagen exhibited poor induction of CYP2B1/2B2 gene expression by PB, monolayer cells on laminin showed substantial induction. The addition of soluble laminin to media did not show any effect on induction in monolayer hepatocytes cultured on TIC. Dishes coated with different concentrations of immovable laminin demonstrated complicated effects. Coating with higher concentrations of laminin resulted in greater induction of CYP2B1/2B2 gene expression by PB. On the other hand, when hepatocytes were cultured on dishes coated with lower concentrations of laminin, they became round and greater induction of CYP2B1/2B2 gene expression by PB was observed. Spherical hepatocytes cultured on low concentrations of TIC also showed highly enhanced induction of CYP2B1/2B2 gene expression by PB. EHS gel overlay to hepatocytes cultured on TIC and collagen sandwich configurations that are known to induce cell polarity enhanced the induction by PB. The induction of CYP2B1/2B2 gene expression needed cytoskeleton organization, such as actin filament, microtubule filament and intermediate filament. These results demonstrate that cell shape, cell density, contact with extracellular matrix and cell polarity all play critical roles in the induction of CYP2B1/2B2 gene expression by PB.

Cytochrome P450 (P450) enzymes are often used in suicide gene cancer therapy strategies to convert an inactive prodrug into its therapeutic active metabolites. However, P450 activity is dependent on electrons supplied by cytochrome P450 reductase (CPR). Since endogenous CPR activity may not be sufficient for optimal P450 activity, the overexpression of additional CPR has been considered to be a valuable approach in gene directed enzyme prodrug therapy (GDEPT). We have analysed a set of cell lines for the effects of CPR on cytochrome P450 isoform 2B1 (CYP2B1) activity. CPR transfected human embryonic kidney 293 (HEK293) cells showed both strong CPR expression in Western blot analysis and 30-fold higher activity in cytochrome c assays as compared to parental HEK293 cells. In contrast, resorufin and 4-hydroxy-ifosfamide assays revealed that CYP2B1 activity was up to 10-fold reduced in CPR/CYP2B1 cotransfected HEK293 cells as compared to cells transfected with the CYP2B1 expression plasmid alone. Determination of ifosfamide-mediated effects on cell viability allowed independent confirmation of the reduction in CYP2B1 activity upon CPR coexpression. Inhibition of CYP2B1 activity by CPR was also observed in CYP2B1/CPR transfected or infected pancreatic tumour cell lines Panc-1 and Pan02, the human breast tumour cell line T47D and the murine embryo fibroblast cell line NIH3T3. A CPR mediated increase in CYP2B1 activity was only observed in the human breast tumour cell line Hs578T. Thus, our data reveal an effect of CPR on CYP2B1 activity dependent on the cell type used and therefore demand a careful evaluation of the therapeutic benefit of combining cytochrome P450 and CPR in respective in vivo models in each individual target tissue to be treated.

In this study, we investigated the combined effects of EGF and collagen I gel on the phenotype of cultured rat hepatocytes and we focussed our investigations on the regulation of xenobiotic-mediated induction of CYP, cell cycle progression and activation of capases 8 and 3. We found that EGF, added to basal culture medium or phenobarbital (3.2 mM) containing medium, provoked a moderate decrease of CYP1A1 and CYP2B1/2 activities. However, EGF did not exert any inhibitory effect on 3-methylcholantrene (5 microM) and beta-naphtoflavone (25 microM) induction of CYP1A1 activities. In collagen gel sandwich cultures, hepatocytes remained arrested in mid-G1 phase of the cell cycle, even in the presence of EGF. In conventional primary cultures, caspases 8 and 3 were activated at 3 and 5 days after plating respectively. In collagen gel sandwich cultures, we found that neither collagen I nor EGF prevented activation of caspase 8 while collagen I gel inhibited activation of caspase 3, preventing spontaneous apoptosis of cultured rat hepatocytes. In contrast, EGF transiently increased caspase 3 activity at day 1 after plating. Altogether, our data demonstrate that collagen I gel triggers intracellular signals which strongly affect cultured hepatocyte phenotype, leading to a cell cycle arrest in G1 phase and long-term survival through the inhibition of caspase 3 activation and that EGF-free medium improves survival and liver-specific gene expression in hepatocytes maintained in collagen I gel sandwich cultures.

In the rat, nicotine is metabolized to cotinine primarily by hepatic cytochrome P450 (CYP) 2B1. This enzyme is also found in other organs such as the lung and the brain. Hepatic nicotine metabolism is unaltered after nicotine exposure; however, nicotine may regulate CYP2B1 in other tissues. We hypothesized that nicotine induces its own metabolism in brain by increasing CYP2B1. Male rats were treated with nicotine (0.0, 0.1, 0.3, or 1.0 mg base/kg in saline) s.c. daily for 7 days. CYP2B1 mRNA and protein were assayed in the brain and liver by reverse transcriptase-polymerase chain reaction (RT-PCR), immunoblotting, and immunocytochemistry. In control rats, CYP2B1 mRNA and protein expression were brain region- and cell-specific. CYP2B1 was not induced in the liver, but CYP2B1 mRNA and protein showed dose-dependent, region- and cell-specific patterns of induction across brain regions. At 1.0 mg nicotine/kg, the largest increase in protein was in the brain stem (5.8-fold, P < 0.05) with a corresponding increase in CYP2B1 mRNA (7.6-fold, P < 0.05). Induction of CYP2B1 was also observed in the frontal cortex, striatum, and olfactory tubercle. Immunocytochemistry showed that induction was restricted principally to neurons. These data indicate that nicotine may alter its own metabolism in the brain through transcriptional regulation, perhaps contributing to central tolerance to the effects of nicotine. CYP2B1 and its human homologue CYP2B6 also activate tobacco smoke procarcinogens such as NNK [4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone]. Highly localized increases in CYP2B could result in increased mutagenesis. These data suggest roles for nicotine-induced CYP2B in central metabolic tolerance, nicotine-induced neurotoxicity, neuroplasticity, and carcinogenesis.

We determined the inducibility, as well as the persistence of the induction, of hepatic microsomal CYP1A1 and CYP1A2 (by western blot analysis), and their catalytic activities (as measured by resorufin ether O -dealkylation) in prepubertal (25-day-old) and adult (120-day-old) offspring of timed-pregnant Sprague–Dawley rats treated with 2,3,7,8-tetrachlorodibenzo- p -dioxin (TCDD). TCDD treatment was subcutaneous, at a low dose of 0.1 μg/kg, on gestational days 7, 14, and 20, and on lactational days 7 and 14. CYP1A1 protein was induced significantly (23-fold) in prepubertal but not in adult offspring of TCDD-exposed dams, whereas ethoxyresorufin O -deethylase (EROD) activity, which is CYP1A1-preferential, was induced less extensively (5-fold) and slightly (1.7-fold) in the prepubertal and adult offspring, respectively. Benzyloxyresorufin O -debenzylase (BROD) activity, which is CYP2B-preferential but has been reported to be catalyzed by CYP1A1, was also induced 5- and 6-fold in prepubertal and adult offspring, respectively, of TCDD-exposed dams. However, the induced BROD activity was neither inhibited by antibody against CYP1A1 nor accompanied by an elevated level of microsomal CYP2B. CYP1A2 was induced slightly only in prepubertal offspring of TCDD-treated dams. There was suggestive evidence of enhanced lipid peroxidation in hepatic microsomes from prepubertal but not adult offspring of TCDD-treated dams. These data showed that in utero plus lactational TCDD exposure effected transient induction of hepatic microsomal CYP1A1 but sustained induction of BROD activity, which may be catalyzed by enzymes other than CYP1A or CYP2B.

The interactions of gender, growth hormone, and phenobarbital induction on Cyp2b expression were examined in phenotypically normal ( lit/+ ) and growth-hormone deficient “little” ( lit/lit ) mice. Using an immunocrossreactive monoclonal antibody designed to identify rat CYP2B1 and 2B2 proteins, we observed three hepatic Cyp2b proteins in control ( lit/+ ) females, but only two proteins, one at trace levels, in control males. Phenobarbital administration to lit/+ mice increased the expression of the two Cyp2b isoforms in the males by 3- to 4-fold, but produced an approximately 75% reduction in the female-expressed proteins. Whereas growth hormone depletion ( lit/lit ) had no effect on the expression profile of Cyp2b proteins in females, it had a de-repressive effect in males, resulting in the expression of three proteins at concentrations now comparable to those observed in female liver. Generally, phenobarbital had no inductive effects in the lit/lit mice of both sexes. In all groups, transcript levels measured by a CYP2B1 probe were in agreement with the protein findings. In contrast, Cyp2b mRNA identified by an oligonucleotide probe for CYP2B2 were repressed completely by growth hormone in both sexes, and was expressed as a female-predominant transcript in the lit/lit mice. In spite of an apparent high degree of sequence homology between the rat CYP2B and murine Cyp2b gene families, the present findings highlight fundamental differences in their constitutive and gender-dependent expression, growth hormone regulation, and phenobarbital inducibility.

Fluvastatin (Fluva), a synthetic inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, induces CYP2B1/2 in rat liver and primary cultured rat hepatocytes. However, the overall profile of CYP induction, which includes induction of CYP4A, suggests that Fluva is not a typical "phenobarbital (PB)-like" inducer. Several treatments affecting diverse cell signaling pathways have been reported to modify PB-inducible CYP2B expression in primary cultured rat hepatocytes. We examined the effects of selected treatments on the ability of Fluva to induce CYP2B1/2 mRNA. Only dexamethasone (Dex) produced effects on Fluva-inducible CYP2B1/2 mRNA expression that differed from those produced on PB-inducible CYP2B1/2 mRNA expression. Dex concentrations up to 10(-7) M of potentiated PB (10(-4) M)-mediated CYP2B1/2 mRNA induction, while higher Dex concentrations produced a progressive reduction in PB-induced CYP2B1/2 mRNA levels. By contrast, Dex concentrations up to 10(-8) M had no effect on Fluva (3 x 10(-5) M)-induced CYP2B1/2 mRNA levels, while Dex concentrations of 10(-7) M and higher markedly suppressed Fluva-mediated CYP2B1/2 mRNA induction. The concentrations of several glucocorticoids that produced suppression of Fluva-induced CYP2B1/2 mRNA levels were the same concentrations that induced CYP3A mRNA. Treatment with pregnenolone 16 alpha-carbonitrile also produced a concentration-dependent suppression of Fluva-induced CYP2B1/2 mRNA levels. Dex-mediated suppression of Fluva-induced CYP2B1/2 mRNA was concentration-dependently reversed when hepatocytes were cotreated with troleandomycin, a selective CYP3A inhibitor. The amounts of Fluva detected in culture medium and cells were reduced significantly when hepatocytes were incubated with Dex. However, Dex-mediated suppression of Fluva-induced CYP2B1/2 mRNA expression was not overcome when hepatocytes were incubated with Fluva concentrations greater than 3 x 10(-5) M, suggesting that mechanisms other than CYP3A-catalyzed metabolism may contribute to Dex-mediated suppression of Fluva-induced CYP2B1/2 expression.

Alligator mississippiensis has at least two classes of inducible hepatic microsomal cytochromes P450 (CYP): (1) those induced by 3-methylcholanthrene (3MC), and (2) those induced by phenobarbital (PB). The rates of induction by these xenobiotic compounds are significantly slower than those reported for mammals. Carbon monoxide binding, western blots, and enzymatic activity measurements indicated that at least 48–72 hr are required to reach full induction. A methoxy-, ethoxy-, pentoxy-, and benzyloxyphenoxazone (resorufin) O-dealkylation (MROD, EROD, PROD, and BROD) profile was indicative of substrate selectivity typical of 3MC- and PB-induced P450s. MROD and BROD showed the greatest ability to discriminate between alligator hepatic microsomes induced by 3MC and PB, respectively. This is in contrast to mammals, in which EROD is a biomarker of polycyclic aromatic hydrocarbon exposure because of its ability to discriminate the induction of CYP 1A. In a similar manner, PROD is a highly preferred activity of CYP 2B in mammals; thus, it is used to indicate CYP 2B induction. The induction of P450 by PB is a general phenomenon in mammals and birds. To the best of our knowledge, this is the first report demonstrating PB induction of P450 activities typical of the mammalian CYP 2 family isoforms in alligator or any reptilian liver. The importance of this finding to the evolution of CYP 2 family regulation by PB is heightened by the fact that induction by this xenobiotic is not common to fish and other lower vertebrates (Ertl RP and Winston GW, Comp Biochem Physiol, in press). Although indicating the presence of CYP 1A- and CYP 2B-like isoforms in alligator, it remains to be established how closely related these alligator P450s are to mammalian isoforms.

Stroke is a neurological condition and may cause changes in hepatic drug-metabolizing enzymes. Hepatic CYP2B is involved in the metabolism of a variety of centrally active substances. The purpose of this study was to investigate the possible down-regulation mechanism of hepatic CYP2B after acute stroke. Using a rat model of acute stroke induced by middle cerebral artery occlusion, we studied the influence of brain ischemia/reperfusion (I/R) injury on CYP2B expression. Effects of 3,5,3'-triiodo-L-thyronine (T3) treatment on constitutive androstane receptor (CAR) and thyroid hormone receptors (TRs, including TRα and TRβ) proteins were detected in Huh7 cells. We found dramatic decreases in the levels of plasma free triiodthyronine, free thyroxine and hepatic CYP2B expression. Both CAR and retinoid X receptor alpha (RXRα) were significantly dissociated from the phenobarbital-responsive enhancer module (PBREM) of the CYP2B1 promoter in the early stages of the acute stroke. The levels of the polymer of TRs, CAR, and RXRα were time-dependently decreased after brain I/R injury. T3 regulated the CAR expression at the transcriptional level, and facilitated the translocation of TRα/β proteins as well as the binding of TRs, RXRα, and CAR to PBREM region. The reduction of thyroid hormone levels after a brain I/R injury may be the initial trigger for the down-regulation of hepatic CYP2B1 via induction of the dissociation of CAR from the TRs and from the PBREM region. Our data suggest that patients with acute ischemic stroke may have a decreased CYP2B-mediated metabolism of exogenous and endogenous compounds because of the low level of thyroid hormones.

The rat CYP2B gene subfamily includes CYP2B1, CYP2B2 and CYP2B3. Translation of an alternatively spliced hepatic CYP2B2 mRNA would generate a CYP2B2 variant, CYP2B2v, having eight additional amino acid residues inserted between CYP2B2 positions 274 and 275. The presence of CYP2B3 and CYP2B2v in rat liver has yet to be demonstrated. cDNA expression vectors were obtained for CYP2B1, CYP2B2, CYP2B3 and CYP2B2v. All four proteins react with an anti-CYP2Bl antibody and can be resolved by SDS-PAGE. A CYP2B3-specific polyclonal antibody raised against an undecapeptide (SPVDPNTIDMT) from near the C-terminus of CYP2B3 detected a constitutive protein on immunoblots of rat liver microsomes, thus demonstrating that the CYP2B3 mRNA is translated in the liver. Similarly, a CYP2B2v-specific polyclonal antibody was raised against a peptide containing the eight additional amino acid residues (VSPAWMRE) predicted to be present in the CYP2B2v protein. It detected a phenobarbital- and Aroclor 1254-inducible protein in rat liver microsomes. Microsomes of Ad293 cells expressing cDNAs for CYP2B2 and CYP2B2v were used to metabolize 7,12-dimethylbenz[a]anthracene (DMBA), and the metabolites produced were compared with those generated by microsomes of cells expressing CYP2B1 cDNA. CYP2B2v had activity similar to that of CYP2B2 for DMBA metabolism. Both CYP2B2 forms preferentially catalyzed 12-hydroxylation, whereas CYP2B1 preferred 7-hydroxylation and exhibited turnover that was strongly suppressed as previously reported. These results demonstrate the existence in rat liver of two new CYP2B proteins: CYP2B3, the major constitutive CYP2B form, and CYP2B2v, which represents a rare case of non-aberrant alternative splicing among xenobiotic-metabolizing P450s.

The dose-response induction of hepatic microsomal pentoxyresorufin O-dealkylase (PROD) activity by phenobarbital (PB) and several polychlorinated biphenyl (PCB) mixtures and congeners was determined in the immature female Sprague-Dawley rat. At a dose of 75 mg/kg/day of PB for 3 days, the microsomal PROD activity was 2154 pmol/min/mg protein. Aroclors 1260, 1254, 1242, and 1016 did not induce maximal PROD activity at doses up to 500 mg/kg, and only Aroclor 1016 induced > a half-maximal response at the 500 mg/kg dose. The relative potencies of eighteen different PCB congeners were also determined, and the structures of these compounds differed with respect to the degree of chlorination (tri- to octochloro) and substitution patterns. The relative potencies of these compounds were estimated by comparing their induced activities at the high dose (150 or 100 mg/kg) with that of PB. The most potent inducers were 2,3,3′,4′,5,6-hexaCB and 2,2′,3,4′,5,5′,6-heptaCB; at a dose of 150 mg/kg, the PROD activity induced by 2,2′,3,4′,5,5′,6-heptaCB was comparable to that observed for PB. 2,3,3′,4′,5,6-HexaCB was the most potent inducer, and hepatic PROD activity in rats treated with 150 mg/kg was 4202 pmol/min/mg; this value was higher than that observed for PB at a dose of 75 mg/kg. A second group of congeners including 2,2′,3,4,4′,5,5′-heptaCB, 2,2′,4,4′,5,5′-hexaCB, 2,2′,3,3′,4,4′,5,5′-oc-taCB, 2,2′,4,4′-tetraCB, 2,2′,4,5,5′-pentaCB, 2,2′,3,4,4′,5′,6-heptaCB, 2,2′,4,4′,5-pentaCB and 2,2′,3,3′,4′,5,5′,6-octaCB induced PROD activity ⩾ 1090 pmol/min/mg at the 150 mg/kg dose, and this value was > 50% of the maximal response observed for PB. The remaining compounds, namely 2,4,4′-triCB, 2,2′,3,4′-tetraCB, 2,2′,5,5′-tetraCB, 2,3′,4,4′,5-pentaCB, 2,3,3′,4,4′-pentaCB, 2,2′,4,4′,5,6′-hexaCB, 2,3,3′,4,4′,5,5′-heptaCB and 2,2′,3,-3′,4,4,5-heptaCB were all relatively weak inducers of hepatic microsomal PROD activity (< 450 pmol/min/mg). In parallel experiments, western blot analysis of immunoreactive CYP2B1 and CYP2B2 proteins showed that PB, the PCB mixtures, and congeners induced both proteins. Previous studies have identified a cis-acting DNA element that plays a role in regulating CYP2B1/B2 gene expression and binds nuclear trans-acting factor(s) induced by PB. The results of gel electrophoretic mobility shift assays with nuclear extracts showed that both PB and 2,2′,3,4′,5,5′,6-heptaCB induce formation of a common retarded band using a 32P-labeled oligonucleotide corresponding to the cis-acting DNA promoter sequence. Both PB and PCBs appear to induce CYP2B1/B2 via a common mechanism. Although the results of this study do not define structure-induction (CYP2B1/B2) relationships for PCBs, two compounds, namely 2,3,3′,4′,5,6-hexaCB and 2,2′,3,4′,5,5′,6-heptaCB, were identified as highly potent inducers.

The O-dealkylations of ethoxyresorufin and pentoxyresorufin are widely used activity probes for measuring the cytochrome P450 forms, CYP1A1 and CYP2B1, respectively, and their induction by xenobiotics, but there is confusion in the literature about which P450 forms are detected in human and rat liver microsomes by these and homologous alkoxyresorufins. High performance liquid chromatographic analysis confirmed that O-dealkylation to resorufin was the sole or predominant route of metabolism for both short-chain and long-chain alkoxyresorufins and benzyloxyresorufin by rat liver microsomes. The purified 3-methylcholanthrene (3MC)-induced rat P450 forms, CYP1A1 and CYP1A2, and a possible variant form, CYP1A1*, showed substrate selectivities for propoxyresorufin, methoxyresorufin and ethoxyresorufin, respectively. Purified phenobarbitone (PB)-induced CYP2B1 was selective for benzyloxyresorufin and pentoxyresorufin. Purified constitutive CYP2C6 was much less active than CYP2B1 or the CYP1A forms but showed distinctive selectivity for benzyloxy-, propoxy- and butoxyresorufin. CYP1A2 and CYP2C6 metabolised n-propoxy- and n-butoxyresorufin much more rapidly (8-23-fold) than iso-propoxy- and iso-butoxyresorufin, whereas CYP1A1 and CYP2B1 showed only small differences (2-5-fold) between the n- and iso-homologues and CYP1A1* and CYP2B2 did not discriminate between them. The results show that ratios between different alkoxyresorufin O-dealkylation (AROD) activities can be more useful than absolute values of single activities for identifying P450 forms. Anti-P450 antibody and furafylline inhibition of rat liver microsomal AROD confirmed that ethoxyresorufin was a selective probe for CYP1A1 in 3MC-induced and isosafrole (ISF)-induced microsomes and that pentoxy- and benzyloxyresorufins both selectively measured CYP2B1 in PB-induced and ISF-induced microsomes. Ethoxyresorufin was not a selective probe for CYP1A in liver microsomes from untreated or PB-induced rats, however, where it was metabolised mainly by CYP2C6 and CYP2B1, respectively. Pentoxyresorufin and benzyloxyresorufin were metabolised by several different P450 forms in non-induced rat liver microsomes but mainly by the CYP1A subfamily in 3MC-induced microsomes and by CYP2B1 in PB- and ISF-induced microsomes. Although with purified rat P450s methoxyresorufin appeared not effectively to discriminate CYP1A2 from CYP1A1, CYP1A1* or CYP2C6, furafylline inhibition indicated that methoxyresorufin was a selective measure of CYP1A2 in uninduced and 3MC-induced rat liver microsomes but not in ISF- or PB-induced microsomes. In human liver microsomes, antibody inhibition and furafylline inhibition showed that ethoxyresorufin and methoxyresorufin were metabolised mainly by CYP1A2, whilst benzyloxyresorufin metabolism was due mainly to the CYP3A subfamily but also involved CYP1A2 and CYP2A6.(ABSTRACT TRUNCATED AT 400 WORDS)

The cytochrome P450 isozyme specificity for the O-dealkylation of methoxyresorufin (MTR) and benzyloxyresorufin (BZR) in the rat and mouse was investigated. The induction of various alkoxyresorufin O-dealkylation activities was measured in male F344/NCr rats exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin or 3,4,5,3',4',5'-hexachlorobiphenyl. MTR and ethoxyresorufin (ETR) O-dealkylation activities were induced 30- and 80-fold, respectively, in the liver. ETR O-dealkylation activity was induced > 250-fold in the kidney, whereas the metabolism of MTR was induced only 30-fold in this extrahepatic tissue. Phenacetin, a fairly specific CYP1A2 inhibitor, caused concentration-dependent competitive inhibition of MTR O-dealkylation (ki approximately 20 microM at 0.5 microM substrate) in hepatic microsomes from 3,4,5,3',4',5'-hexachlorobiphenyl-treated rats. The corresponding ki for inhibition of ETR O-dealkylation by phenacetin was > or = 333 microM at a 0.5 microM substrate concentration. A monoclonal antibody displaying inhibitory activity against rat CYP1A1 inhibited ETR O-dealkylation activity, whereas it failed to inhibit MTR O-dealkylation activity. In contrast, a monoclonal antibody reactive with both CYP1A1 and CYP1A2 inhibited both O-dealkylation activities to an equal extent. Similar experiments, employing phenacetin or specific monoclonal antibodies, yielded comparable results when performed with mouse microsomes. The maximal induction of MTR O-dealkylation activity in mice was > 100-fold. The P450 isozyme specificity of BZR O-dealkylation was also examined in both rats and mice. Pregnenolone-alpha-carbonitrile, a strong inducer of CYP3A, only weakly induced BZR O-dealkylation activity. In addition, a monoclonal antibody that specifically inhibits CYP2B caused inhibition of BZR metabolism in microsomes from phenobarbital- or dexamethasone-pretreated rats. In B6C3F1 mice exposed to dietary Aroclor 1254, significant induction of hepatic MTR O-dealkylation activity was observed at concentrations lower than those required for the induction of ETR or BZR O-dealkylation. In summary, it would appear that MTR is a relatively specific substrate for CYP1A2 activity in rodents, while BZR appears to be relatively specific for CYP2B.

The effect of a nitrogen heterocycle constituent on the ability of arylmethanes to induce phase I and phase II drug-metabolizing enzymes has been examined. Rats were treated with tetra-, tri-, di- or monoarylmethane compounds daily for 3 days at a dose of 75mg/kg. Induction of UDP- glucuronosyltransferase(morphine) activity was seen with twelve of the eighteen compounds investigated and for three compounds it occurred independent of any induction of cytochrome P450. Induction of glutathione S -transferase activity was seen with ten of the compounds and was generally paralleled by changes in overall cytochrome P450 concentration and in both pentoxyresorufin and erythromycin dealkylase activities. Major induction of ethoxyresorufin deethylase activity was only apparent with two diarylmethanes that contained a 1-substituted imidazole moiety. UDP-glucuronosyltransferase(1- naphthol) activity was coinduced by these two compounds. A tthird compound, diphenyl-4-pyridylmethane induced UDP-glucuronosyltransferase(1-naphthol) activity without increasing ethoxy-resorufin deethylase activity. Cytosolic sulfotransferase activity was not induced by the administration of any compound in this study. Among arylmethane derivatives, the presence of two aryl groups appeared to be a minimum requirement for induction of drug-metabolizing enzymes. If one of the aryl groups was not a heterocycle, or if the nitrogen atom of the heterocycle was sterically hindred, major induction of cytochrome P450 did not occur. With triarylmethanes, induction was independent of whether the heterocycle was imidazole, pyridine or pyrimidine.

Cytochrome P450 isozymes induced in rat liver by a range of concentrations of toluene were studied with monoclonal antibodies (MAbs) to specific P450 isozymes and by enzyme assays. Nitrosodimethylamine demethylase activity was significantly increased in microsomes from rats exposed to more than 1000 ppm of toluene, an increase that was dose-dependent. Anti-CYP2E1 significantly inhibited the metabolism of toluene to benzyl alcohol (BA) by about 50%, in microsomes from 1000 to 4000 ppm toluene-exposed rats, at low substrate concentration (0.2 mM). With anti-CYP2Bl/2, the rate of BA formation was decreased by 15–17% in microsomes from rats of 2000 and 4000 ppm toluene exposures at high substrate concentration (5.0 mM). On the other hand, anti-CYP2C11/6 inhibited the rate of formation of BA in all of the microsomes, but the extent of inhibition was progressively decreased from 55% in control to 33% in 4000 ppm exposure. Immunoblot analysis with anti-CYP2E1 and anti-CYP2B1/2 revealed stronger immunoreactive bands in microsomes from rats exposed to more than 1000 and 2000 ppm of toluene, respectively. Stronger bands were also observed in microsomes from rats of 2000–4000 ppm toluene exposures with anti-CYP3A1/2, but no immunoreactivity appeared with anti-CYP1A1/2. These results suggest that toluene induces CYP2E1, CYP2B1/2 and CYP3A1/2, but reduces CYP2C11/6, and has no effect on CYP1A1/2.

We have analysed the induction of microsomal and peroxisomal proteins and their RNAs after treatment of hepatocytes with the peroxisome proliferator, clofibric acid, in vitro and in vivo. After treatment of hepatocytes with 1 mM clofibric acid for 4 days, P450 4A1 RNA is induced 500-fold, and acyl-CoA oxidase and P450 2B1 280-fold, relative to control cultures. These RNAs are detectably induced after administration of 25 microM clofibric acid, and show a similar induction response with increasing doses of clofibric acid. Western blot analysis of the P450 4A and bifunctional enzyme (BFE) proteins showed that both were induced in parallel with increasing doses of clofibric acid, over a range of 25 microM-1 mM. The distribution of the induced proteins was examined by immunocytochemistry. Increasing doses of clofibric acid led to an increase in the average intensity of staining for both proteins throughout the hepatocyte population. There was, however, a graded variation between hepatocytes in the intensity of staining, both for P450 4A and BFE proteins. The heterogeneity in response of the hepatocyte population in vitro may be related to differential sensitivity of hepatocytes to induction in vivo. Therefore, rats were dosed with 0, 50 or 300 mg/kg of clofibric acid for 4 days by gavage, and the livers were examined by immunocytochemistry. After 50 mg/kg of clofibric acid, both P450 4A and BFE were induced mainly in zones 3 and 2 of the liver acinus. However, after 300 mg/kg of clofibric acid, staining for both proteins was strong and homogenous throughout the liver acinus. Thus, hepatocytes from zones 3 and 2 of the acinus are differentially responsive to induction by clofibric acid.

We studied the subcellular distribution of cytochrome P450 and related monooxygenase activities in six regions of human brains removed at autopsy. The content of total cytochrome P450 was found to be at least nine times higher in the mitochondrial fraction than in the microsomes in all the regions studied. However, cytochrome P450-dependent enzymatic activities which are representative of different isoforms metabolizing exogenous molecules exhibited a microsomal prevalence, a situation previously observed in rat brain. The other drug-metabolizing enzymes catalysing functionalization and conjugation reactions, presented the following characteristics in human brain: (i) a low activity of NADPH-cytochrome P450 reductase, which also catalyses the reduction of some xenobiotics; (ii) a high specific activity of the membrane-bound epoxide hydrolase; (iii) among the enzymes catalysing conjugation reactions, 1-naphthol-UDP-glucuronosyltransferase activity was barely or not detectable, whereas the mean glutathione-S-transferase activity was 15 times higher than the activity measured in rat brain. The presence of several drug-metabolizing enzyme activities in human brain microvessels, and particularly the high activity of epoxide hydrolase, suggests a participation of these enzymes in the metabolic blood-brain barrier.

Phenothiazine tranquilizers have been associated with pharmacokinetic drug interactions in man. In this study the in vivo and in vitro effects of the clinically important phenothiazines promazine (PZ) and chlorpromazine (CPZ) on drug oxidations catalysed by specific cytochrome P450 (P450) enzymes were investigated in the rat. In vitro , the two drugs were relatively ineffective inhibitors of constitutive P450 activities, but were inhibitory toward the principal phenobarbital-inducible P450 2B1 and, to a lesser extent, P450 1A1. Administration of PZ and CPZ to male rats did not markedly influence the total microsomal P450 content of the liver. However, the quantitatively important male-specific P450 2C11 was down-regulated by CPZ and concomitant induction of P450 2B1 and associated 7-pentylresorufin O -depentylase activity were noted. A small increase in the activity of microsomal 7-ethylresorufin O -deethylase was also observed following administration of both drugs to rats, suggesting induction of P4501A1/2. Considered together, it is apparent that the two phenothiazines are preferential inhibitors and inducers of P450 2B1 in rat liver. Drug interactions in humans involving phenothiazines may reflect a combined effect of induction and inhibition processes as well as down-regulation of other P450s, such as that produced by CPZ on P450 2C11.

Intravenous treatment of male rats with recombinant human interleukin-6 (rhIL6) at 50, 100 and 200 micrograms/kg (corresponding to 4, 8 and 16 x 10(4) U/animal, respectively) reduced the activities of hepatic microsomal cytochrome P450-dependent monoxygenases to varying degrees. Ethylmorphine-N-demethylase activity fell to 53% of control values, an effect similar to that induced by 2.5 mg/kg Escherichia coli lipopolysaccharide (LPS). Ethoxycoumarin-O-deethylase activity was also sensitive to inhibition, whereas IL6 had little effect on the activities of other P450-dependent enzymes, including ethoxyresorufin-O-deethylase. Pentoxyresorufin dealkylase activity, which is representative of the cytochrome P450 IIB 1/2 subfamily, was unaffected by IL6 whereas LPS reduced it to 33.7% of control values. Another hepatocyte-related parameter, serum concentration of alpha 1-acid glycoprotein (AGP), was increased by up to 3.5-fold over baseline by IL6 and 10-fold by LPS. Recombinant human interleukin-1 beta (rhIL1 beta) (10 micrograms/kg, corresponding to 5 x 10(4) U/rat) and recombinant human tumor necrosis factor alpha (rhTNF) (150 micrograms/kg corresponding to 24 x 10(4) U/rat) were both as potent as LPS (2.5 mg/kg) in increasing serum AGP levels and reducing hepatic microsomal monoxygenase activities. IL6 did not potentiate the effects of rhIL1 beta. Hepatic microsomal glucuronyltransferase activities were little affected by LPS and unaffected by rhIL6. Finally, rhIL6 was more potent after i.p. injection than after i.v. or s.c. injection. These results suggest that the effects of LPS, TNF and IL1 on the mixed-function oxidase system in vivo may be due partly to an induction of IL6 in vivo. The different sensitivities of the enzymes to IL6 but not to IL1 or TNF may be due to the involvement of two distinct mechanisms.

This study was undertaken to investigate the effect of dietary lipid on the regulation of several constitutive P450 isozymes. Male Sprague-Dawley rats with body weights of 130–140 g were fed either a 20% corn oil (CO) diet or a fat-free (FF) diet for 4 days following 2 days of fasting. Using liver microsomes, the catalytic activities and immunochemically detectable protein levels of P450s 1A1 and 2, 2A1, 2B1 and 2, 2C11, 2E1, and 3A were determined. The microsomes from rats fed the 20% CO diet exhibited 2-fold higher levels in N -nitrosodimethylamine demethylase activity and P450 2E1 protein than those from rats fed the FF diet. the CO group also showed 2.5-fold higher levels in 6β-hydroxylation of testosterone and P450 3A protein than the FF group. In contrast, the CO diet did not affect the immunodetectable level of P450 2C11 protein and its catalytic activities such as benzphetamine demethylase activity and 2α-hydroxylation of testosterone. P450 1A1 was not detectable in either group, but 1A2 was 2.5-fold higher in the CO group than in the FF group. In the liver, the P450 2B1 level was very low in both groups as measured by pentoxyresorufin dealkylase activity and the protein level, whereas 2B2 was 2.5-fold higher in the CO diet group. In lung microsomes from rats fed different amounts of CO, an inverse relationship was observed between the P450 2B1 level and the dietary CO level. the results suggest that the constitutive levels of P450 isozymes are modulateted by dietary lipid in a selective manner; the levels of hepatic P450s 1A2, 2B2, 2E1, and 3A were regulated positively but the level of pulmonary P450 2B1 was suppressed by dietary lipid.

Phenobarbital (PB) and certain structurally-related compounds induce a variety of hepatic drug-metabolizing enzymes in many strains of rats. Thus, following administration of PB (300, 500 ppm), barbital (BB, 1500 ppm) or 5-ethyl-5-phenylhydantoin (EPH, 500 ppm), CYP2B1-mediated benzyloxyresorufin O-dealkylase activity and epoxide hydrolase activity were profoundly induced in female DA and F344/NCr rats. In contrast, outbred female lean and obese Zucker rats showed markedly reduced CYP2B1 responses (less than 15% and less than 5% of those observed in the female DA or F344/NCr rat) to PB (doses less than or equal to 300 ppm), BB (1500 ppm) or EPH (500 ppm). In parallel studies, profound increases in RNA levels coding for CYP2B1, glutathione S-transferases Ya/Yc (alpha subclass), or epoxide hydrolase were detected in the female F344/NCr rat following treatment with PB (300 ppm), BB (1500 ppm) or EPH (500 ppm). In contrast, lean Zucker rats showed a strong response only to the highest dose of PB (500 ppm), implying that the diminished response in the Zucker rats may occur at some pretranslational level. Similar studies with lower doses of PB, EPH or BB in male lean Zucker rats showed a decreased response, relative to that in male F344/NCr rats. However, this insensitivity was not as profound as that observed in the female Zucker rats. In fact, the response to PB-type inducers in male or female Zucker rats is probably most clearly explained as a shift of the dose-response curve sharply to the right (decreased responsiveness, compared to F344/NCr or DA rats of the same sex). This decreased responsiveness of female lean Zucker rats to induction of CYP2B1, relative to that of F344/NCr rats, was also observed with the structurally-diverse PB-type inducers clonazepam, clotrimazole and 2-hexanone. In contrast, the female Zucker rat (obese or lean) displayed a pronounced response to induction of CYP1A-mediated ethoxyresorufin O-deethylase activity by beta-naphthoflavone, a prototype inducer of CYP1A1 and CYP1A2. The Zucker rat would thus appear to represent a potentially exploitable genetic model for examining the mechanism of enzyme induction by the myriad xenobiotics which induce a PB-type response.

A hepatic cell line has been immortalized after simian vacuolating virus 40 infection of adult rat hepatocytes maintained in defined culture conditions. This cell line, designated SVHep B4, expressed nuclear large T antigen, exhibited an extended lifespan (50 subcultures) and had a hepatocyte-like morphology. Expression and regulation of drug metabolizing enzymes were studied in long-term cultures of SVHep B4 cells. Significant activities of phase I and phase II enzymes were detected. gamma-Glutamyltransferase, a marker often increased in neoplastic and dedifferentiated hepatocytes, showed a low activity whereas the hepatospecific enzyme tyrosine aminotransferase was expressed at levels similar to those in liver. Responsiveness of drug metabolizing enzymes to inducers was investigated with phenobarbital, dexamethasone and methylcholanthrene. IIB and IA subfamilies of cytochrome P450 were increased, respectively, by phenobarbital (170%) and methylcholanthrene (500%). Glucuronidation of 1-naphthol was increased by phenobarbital (140%) and 3-methylcholanthrene (160%). Phenobarbital, methylcholanthrene and dexamethasone were found to increase significantly gamma-glutamyltransferase while tyrosine aminotransferase activity was enhanced by dexamethasone. Stable expression and inducibility of drug metabolizing enzymes in long-term cultures of the SVHep B4 cell line demonstrate that immortalization of adult hepatocytes represents a promising tool for drug biotransformation studies in vitro.

The effects of treatment with phenobarbital, 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP), pregnenolone-16 alpha-carbonitrile (PCN), 3-methylcholanthrene (3-MC) and isosafrole on the hepatic microsomal formation of nine monohydroxy metabolites of testosterone and the O-dealkylation of the ethyl and pentyl ethers of resourfin were evaluated in adult male C57BL/6J and DBA/2NCR mice. In both strains, phenobarbital, TCPOBOP and PCN induced testosterone 2 beta-, 6 beta-, 15 beta- and 16 beta-hydroxylases up to 5-fold, while phenobarbital and TCPOBOP increased the rate of dealkylation of pentoxyresorufin by approximately 30-fold. However, phenobarbital and TCPOBOP did not exhibit identical patterns of induction for the testosterone oxidation reactions. Hepatic microsomes from C57BL/6J mice treated with TCPOBOP displayed a depression in 6 alpha-testosterone hydroxylase activity, which was also observed in PCN-treated animals, whereas phenobarbital-treated mice exhibited an elevation in this monooxygenase activity. A dose of TCPOBOP (0.5 mumol/kg) previously demonstrated to represent an ED50 for mouse aminopyrine N-demethylase activity was also found to approximate the ED50 for pentoxyresorufin O-dealkylase activity in the C57BL/6J mouse. Isosafrole or 3-MC treatment had little effect on testosterone metabolism or pentoxyresorufin O-dealkylase activity in either strain, while 3-MC induced ethoxyresorufin O-deethylase activity in C57BL/6J but not DBA/2NCR mice. This study confirms that TCPOBOP is a potent cytochrome P-450 inducer which most closely resembles phenobarbital in its mode of action. However, TCPOBOP and phenobarbital do not evoke identical modulations of cytochrome P-450-dependent monooxygenases in mice.

Multiple hepatic cytochrome P450 enzymes are down-regulated at the mRNA and protein levels during inflammation and infection. A body of evidence suggests that nitric oxide (NO) produced from inducible NO synthase (NOS2) is responsible for some of these effects. The current study was designed to examine the NO dependencies of the down-regulation of phenobarbital-induced CYP2B mRNAs and proteins by bacterial endotoxin (lipopolysaccharide, LPS) treatment in vivo, using an NOS2-null mouse model. Treatment of C57/BL6 mice with 0.3 mg/kg of LPS maximally suppressed phenobarbital-induced CYP2B9 and 2B10 mRNAs measured 12 hr after injection, whereas 1-10 mg/kg of LPS was required to elevate NO production. Down-regulation of CYP2B mRNAs by 1 mg/kg of LPS was equivalent in wild-type and NOS2-null mice. No effect of LPS in the dose range of 0.3 to 10 mg/kg was observed on microsomal CYP2B protein levels measured 12 hr after treatment, whereas 1 mg/kg of LPS suppressed CYP2B proteins 24 hr after treatment in both wild-type and NOS2-null mice. We conclude that the main mechanism for the down-regulation of CYP2B proteins in mouse liver following moderate- or high-dose LPS treatment is via NO-independent suppression of CYP2B9 and 2B10 mRNAs. Unlike rat hepatocytes, the contribution of a rapid, NO-dependent mechanism of CYP2B protein suppression in mouse liver appears to be minor or non-existent.

Multidrug resistance-associated protein 2 (MRP2) is a drug efflux pump found at the biliary pole of hepatocytes. In the present study, we have investigated its expression in response to phenobarbital, a liver tumor promoter known to up-regulate hepatic cytochromes P450 (CYPs), such as CYP2B1/2 and CYP3A1/2. MRP2 mRNA and protein levels were found to be markedly increased in both primary rat and human hepatocytes exposed to phenobarbital. However, features of this up-regulation, especially the dose-response, were different from those of the induction of CYP2B1/2 and CYP3A1/2. In addition, hepatic MRP2 expression remained unaltered in rats treated by phenobarbital that, by contrast, increased CYP2B1/2 and CYP3A1/2 gene expression in the liver. Therefore, MRP2 and CYPs appeared differently regulated in response to phenobarbital in both in vivo and in vitro situations, suggesting that cellular and molecular mechanisms underlying up-regulation of MRP2 are, at least in part, unrelated to those operating for CYPs. Phenobarbital-related MRP2 induction in primary rat hepatocytes was associated with some phenotypic effects of the barbiturate, such as prolonged cell survival and inhibition of cell proliferation. Phenobarbital also inhibited growth of human hepatoma HepG 2 cells and increased their level of MRP2 gene expression. Such results may favor a putative relationship between phenobarbital-mediated MRP2 regulation in cultured liver parenchymal cells and alteration of cell cycle and survival.

A higher proportion of alcoholics than non-alcoholics smoke (80 vs 30%). In animals, chronic administration of alcohol induces tolerance to some effects of nicotine. To investigate if chronic ethanol (EtOH) induces alterations in CYP2B1/2 and nicotine C-oxidation activity, male rats (N = 4-6/group) were treated once daily with saline or EtOH (0.3, 1.0, and 3.0 g/kg, p.o./by gavage) for 7 days. A quantitative immunoblotting assay was developed to detect CYP2B1/2 in the brain, where constitutive expression is low, and in the liver. Using this method, it was determined that EtOH did not alter CYP2B1/2 protein expression significantly in six brain regions (olfactory bulbs, olfactory tubercles, frontal cortex, hippocampus, cerebellum, and brainstem). However, a dose-dependent induction of CYP2B1/2 protein expression was detected in the liver. Significant induction of 2-, 3-, and 2.7-fold were observed for the 0.3, 1.0, and 3.0 g/kg doses, respectively. Increases were also observed in CYP2B1 mRNA, which was induced by 14, 38, and 43% at the same doses. Liver microsomal nicotine C-oxidation also was increased (1.3 to 4.5-fold). CYP2B selective inactivators demonstrated that approximately 70% of nicotine C-oxidation was mediated by CYP2B1/2 in both EtOH-induced and uninduced hepatic microsomes. In summary, chronic, behaviorally relevant doses of EtOH induce CYP2B1/2 protein, mRNA, and nicotine C-oxidation activity in rat liver but not in rat brain, and these increases could contribute to cross-tolerance and co-abuse of ethanol and nicotine.

Numerous xenobiotics are capable of inducing their own metabolism and by enzyme induction can also lead to enhanced biotransformation of other xenobiotics. In this project, we examined the influence of pyrethroids (permethrin, cypermethrin, and fenvalerate) on the expression and activity of the phenobarbital (PB)-inducible cytochrome P450 2B1 isoform (CYP2B1) in primary rat hepatocyte cultures. Incubation of hepatocyte cultures with pyrethroids resulted in a marked CYP2B1 induction. Among the tested pyrethroids, permethrin elicited the most pronounced induction of CYP2B1 mRNA, which exceeded maximal induction achieved by PB at concentrations approximately 10-fold higher. Furthermore, permethrin induced CYP3A1 mRNA expression, while the expression of the CYP1A1 isoform, which in vivo is not responsive to PB treatment, was not significantly affected by pyrethroids. Permethrin-dependent enhancement of CYP2B1 and CYP3A1 mRNA expression was repressed by the hepatotrophic cytokine epidermal growth factor, which is known to also inhibit PB-dependent induction of CYP2B1. Several metabolites of permethrin formed by hepatocytes (3-(2',2'-dichlorovinyl)-2,2-dimethylcyclopropanecarboxylic acid, 3-phenoxybenzyl alcohol, and 3-phenoxybenzoic acid) were ineffective in inducing CYP2B1 mRNA. Furthermore, permethrin stimulated the expression of the luciferase reporter gene under control of the CYP2B1 promoter (comprising the PB-responsive enhancer module) in transiently transfected primary hepatocyte cultures. Thus, permethrin-stimulated gene expression occurred on the transcriptional level. Taken together, these results indicate that the pyrethroid permethrin is a PB-like inducer. Due to its superior potency in induction, permethrin appears as a useful substance for mechanistic studies to elucidate the mechanism of enzyme induction by phenobarbital.

This study was designed to test the hypothesis that the reduction in cytochrome P450 (CYP) 2B1 content and activity of rat lung microsomes, following dosing with pneumotoxic trimethylphosphorothioates, results from damage to specific cell types. Of the lung cells exhibiting immunolabelling for CYP2B1, only type I cells showed signs of susceptibility to the pneumotoxins O,O.S-trimethylphosphorothioate and O,S,S-trimethylphosphorodithioate. While most type I cells became necrotic, type II and Clara cells showed no signs of injury, despite their gradual loss of CYP2B1, as detected by immunogold labelling. This loss of labelling was accompanied by a 75% reduction in the immunoreactive CYP2B1 content and an 85% reduction in pentoxyresorufin O-dealkylase activity in lung microsomes. In contrast, the non-pneumotoxic analogue O,O,S-trimethylphosphorodithioate, differing from O,O,S-trimethylphosphorothioate by only the presence of a P = S rather than a P = O moiety, caused an even more rapid fall in pulmonary pentoxyresorufin O-dealkylase activity, but only a slight reduction in the microsomal content of CYP2B1. The recovery of this activity began within 12 hr of dosing. O,O,S-Trimethylphosphorodithioate, which acts as a suicidal inhibitor of pulmonary CYP2B1, did not cause any detectable lung injury or increase in cell division. These results are consistent with the initial reduction in both enzyme content and activity caused by the P = O - containing pneumotoxins resulting, almost entirely, from death of type I cells. Subsequent reductions that occur long after clearance of the toxin may be exacerbated by the onset of mitosis in Clara and type II cells.

Phenobarbital (PB) was shown to induce the major PB-inducible cytochrome P450 (CYP) isoforms, CYP2B1/2, in perivenular hepatocytes by a single injection, and in midzonal and periportal hepatocytes in addition to perivenular hepatocytes by injections of the same dosage once a day for 3 days in rat livers. The present study was undertaken to determine whether the spread of enzyme induction to midzonal and periportal hepatocytes is caused by the increase in total dose of the drug by repetitive injections or by the repetitive injections of the drug themselves. Male adult rats were administered PB by a single injection (80 mg/kg) or repetitive injections (20 mg/kg once a day for 4 days; a total dose of 80 mg/kg), and the molar content of CYP2B1/2 was measured by quantitative immunohistochemistry in the cytoplasm of perivenular, midzonal, and periportal hepatocytes. In addition, the molar content of total CYP in the cytoplasm was measured by microphotometry, and the expression of CYP2B2 mRNA was examined by in situ hybridization. When animals received the single injection, the isoforms and CYP2B2 mRNA increased markedly in perivenular hepatocytes, increased somewhat in midzonal hepatocytes, and remained unchanged in periportal hepatocytes. If animals received the repetitive injections, however, although the isoforms and the mRNA increased markedly in perivenular hepatocytes, they also increased markedly in midzonal hepatocytes and somewhat in periportal hepatocytes. These findings demonstrated that the enlargement of the sublobular area in which induction of the isoforms occurred was caused by the repetitive injections of PB themselves.

The metabolism of toluene in human liver microsomes and by cDNA-expressed human cytochrome P450s (CYPs) was investigated. Toluene was metabolized mainly to benzyl alcohol and slightly to o- and p-cresol by human liver microsomes. Formation of o-cresol was elevated in microsomes from human livers derived from cigarette smokers, but the induced CYP isoforms were not clear. Of the eleven human CYP forms studied, CYP2E1 was the most active in forming benzyl alcohol, followed by CYP2B6, CYP2C8, CYP1A2, and CYP1A1, in that order. The activities of CYP2A6, CYP2C9, CYP2D6, CYP3A3, CYP3A4, and CYP3A5 were negligible. In addition, CYP2B6 and CYP2E1 catalyzed the formation of p-cresol (11-12% of total metabolites), and CYP1A2 catalyzed the formation of both o-(22%) and p-cresol (35%). The relationship between the amino acid sequence of rat CYP2B1 cDNA and the activity for toluene metabolism was investigated using variants, because of great differences in the forming of toluene ring products between CYP2B1 and CYP2B6. These results suggest that the structure of CYP2B1 at the site of Leu 58 rather than Ile-114 and Glu-282 plays an important role in the formation of toluene ring products, whereas in CYP2B1 Ile-114 plays an important role in the formation of benzyl alcohol. These results may explain, in part, the lower activity of CYP2B6, which has Phe at position 58 of the protein, for toluene ring oxidations than that of CYP2B1.

The expression of cytochromes P450 2E1, P450 2B and P450 1A was examined in rat hepatic tissue in response to YH439, an experimental hepatoprotective agent. P450 2E1 metabolic activities relatively specific for P450 2E1 were decreased up to 57% of control activities in the hepatic microsomes prepared from rats treated with YH439 for 3 days. Immunoblot analyses showed that P450 2E1 levels were decreased below the limit of detectability in hepatic microsomes prepared from YH439-treated rats. YH439 at doses from 25 to 100 mg/kg completely suppressed isoniazid-inducible P450 2E1 levels as monitored by both metabolic activities and immunoblot analysis. RNA hybridization analysis revealed that P450 2E1 mRNA levels failed to change after YH439 treatment. These results demonstrate the YH439 effectively suppresses P450 2E1 expression in the absence of transcriptional inactivation. YH439 failed to affect P450 2B1/2 expression, whereas this agent enhanced the hepatic P450 1A1/2 levels. The hepatoprotective effects of YH439 were also examined. Animals treated with CC1 4 and ethanol for 9 weeks showed hepatic injury as demonstrated by 2.5- and 2-fold increases in serum alanine aminotransferase and alkaline phosphatase activities, respectively. Concomitant YH439 treatment resulted in a significant protective effect against the experimental hepatic injury. The toxicant-induced elevation in hepatic hydroxyproline level was completely blocked by YH439 treatment. These data indicate that YH439 suppresses the expression of P450 2E1 and protects the liver against chemical-induced hepatic injury and that the selective modulation of detoxifying enzymes by YH439 may contribute to the protection of liver from xenobiotic-induced intoxication.

Phenobarbital-dependent induction of mouse cytochrome P-450 (Cyp) orthologous to rat CYP2B1 and its modulation by hepatotrophic growth factors were examined in primary hepatocyte cultures. Compared to rat hepatocytes, induction in mouse hepatocytes was more rapid and effective. Ligands of the EGF receptor, epidermal growth factor, and transforming growth factor alpha inhibited induction on the basis of protein expression and CYP2B-associated 7-pentoxyresorufin-O-depentylase activity. Furthermore, EGF led to repression of accumulation of corresponding mRNA under phenobarbital, an effect not blocked by inhibition of protein synthesis under cycloheximide. Ligands of the EGF receptor may contribute towards the decrease in hepatic CYP expression observed during (pre)neoplastic development and regeneration.

This study describes the effects of chronic ethanol (ETOH) treatment and withdrawal on the rat hepatic mixed-function mono-oxygenase system. Male Sprague-Dawley rats (150-200 g, 10 per group) were administered ETOH as part of the Lieber-deCarli liquid diet for 3 weeks. Ethanol was removed, and the animals were euthanized at 0, 24, 48, 72 and 168 hr post-withdrawal. Microsomes were prepared, and ethanol-inducible cytochrome P4502E1 (CYP2E1) activity was measured using the enzyme markers N-nitrosodimethylamine demethylase (NDMAd), p-nitrophenol hydroxylase (PNPH) and aniline hydroxylase (AH). Activities were found to be induced significantly after chronic ETOH feeding using all three assays (NDMAd, 5-fold; PNPH, 3.5-fold; AH, 9-fold). Upon ETOH withdrawal, all three activities dropped markedly, with NDMAd and PNPH at control values at 24 hr and all subsequent time points. AH activity remained 3-fold higher than controls at 24, 48 and 72 hr. Western blot analyses showed that immunoreactive CYP2E1 returned to control at 24 hr, consonant with NDMAd and PNPH activities. The prolonged induction of AH activity following ETOH withdrawal indicates that it is not a specific marker of CYP2E1-catalyzed reactions. Collectively, these data are suggestive of a rapid mechanism of CYP2E1 degradation in the rat liver. Of the other parameters investigated in this study, total cytochrome P450 content was increased 2.5-fold after ETOH feeding, with levels dropping markedly 24 hr post-withdrawal. NADPH-dependent cytochrome c reductase activity was unchanged throughout the course of the study. CYP1A1, CYP2B1 and CYP3A activities were assessed by the substrate probes ethoxyresorufin O-dealkylase (EROD), pentoxyresorufin O-dealkylase (PROD) and erythromycin N-demethylase (ERNd). EROD and PROD were induced significantly by ETOH administration (2-fold) at 0 hr, with EROD remaining elevated over controls 24 hr post-withdrawal. Quantitative western blot analysis of CYP1A1 and CYP2B1 revealed a pattern of immunostaining generally consistent with but less variable than levels predicted by the respective substrate markers. Both proteins were induced significantly by chronic ethanol administration (CYP1A1, 1.9-fold; CYP2B1, 4-fold). Induction of these P450 isoforms persisted for several days following withdrawal. In contrast, immunoreactive CYP1A2 was found to decrease significantly (by 30-40%) during ethanol withdrawal (24, 48, 72, 168 hr). ERNd activity was induced significantly by chronic ETOH feeding (2.5-fold) and remained so for 24 hr into the withdrawal period (2-fold). Immunoreactive CYP3A1 was also induced significantly following ETOH administration (0 hr) and 24 hr following withdrawal.(ABSTRACT TRUNCATED AT 400 WORDS)

Cultured rat hepatocytes have been used to compare the relative activities of cytokines to inhibit the phenobarbital (PB) or 3-methylcholanthrene (MC) induction of cytochrome P4502B1 and 2B2 (P4502B1/2) or P4501A1 and 1A2 (P4501A1/2), respectively. Recombinant cytokines tested were human interleukin-6 (IL-6), interleukin-1 alpha and -beta (IL-1 alpha and IL-1 beta, respectively), and rat gamma-interferon (INF gamma). Hepatocytes were cultured in the presence of 2 mM PB or 1 microgram MC/mL culture medium for 24 hr with or without the cytokines. Benzyloxyresorufin and ethoxyresorufin O-dealkylase (BROD and EROD, respectively) activities were determined as indices of P4502B1/2 and P4501A1/2, respectively. All cytokines produced a concentration-dependent inhibition of the PB induction of BROD activity. IL-1 beta and IL-6 were approximately equipotent with IC50 values of 1-2 U/mL, causing greater than 90% inhibition of PB induction of BROD activity at a concentration of 50 U/mL culture medium. IL-1 alpha tended to be less active. PB induction of BROD activity was also inhibited by INF gamma, but higher concentrations (62.5 to 500 U/mL culture medium) were required. All cytokines were less effective in inhibiting the MC induction of EROD activity than the PB induction of BROD activity. IL-1 beta and IL-6, at 50 U/mL culture medium, inhibited EROD induction by only 35% compared with the greater than 90% inhibitory effect on the PB induction of BROD activity. INF gamma was ineffective in inhibiting EROD activity at the concentrations studied. Western immunoblot analysis indicated that the cytokines prevented the ability of the inducers to increase the expression of P4502B1/2 and P4501A1/2 immunoreactive proteins, and this effect correlated with their inhibitory effect on induction of enzyme activity. The results suggest that inducible isoforms of cytochrome P450 differ in their susceptibility to regulation by the cytokines, and that cytokines possess differential activity to inhibit the induction of P450 isoforms, with IL-1 beta and IL-6 being the most effective.

Exposure to benzene was reported to lower the cytochrome P450 (CYP; EC 1.14.14.1) content in phenobarbital-pretreated (PB) rats in vivo (Gut I, Zbl Pharm 122: 1139-1161, 1983). In this paper we followed the ability of benzene and its metabolites, phenol, catechol, hydroquinone and benzoquinone to destroy CYP in liver microsomes from PB rats in vitro. The spectrophotometric determinations of the total CYP content, 7-pentoxyresorufin O-depentylase and aniline hydroxylase activities, electrophoresis and western blot analysis after incubation of PB-microsomes with benzene or its metabolites revealed that: (1) benzene is metabolically activated to intermediates causing CYP destruction; phenol is not responsible for this effect. (2) Quinonic metabolites of benzene cause CYP destruction with different potency (30% CYP was destroyed by 3 mM catechol, 0.3 mM hydroquinone and 0.03 mM benzoquinone). (3) Low concentrations of quinones are capable of protecting CYP against reactive oxygen species produced in the CYP futile cycle. (4) Ascorbate effectively protects CYP against quinones, apparently by maintaining them in the reduced state. (5) Quinones attack both heme and protein of CYP. (6) CYP activities differ in the sensitivity to quinone-mediated destruction. In conclusion, we suggest that quinones may be responsible for CYP destruction by benzene in vivo.

The expression of constitutive and inducible cytochrome P450 forms was measured in cynomolgus monkey liver and compared with man, rat, mouse and hamster. Four alkoxyresorufin O-dealkylation (AROD) activities widely used as indicators of P450 induction were measured: methoxyresorufin O-demethylation (MROD), ethoxyresorufin O-deethylation (EROD), pentoxyresorufin O-dealkylation (PROD) and benzyloxyresorufin O-dealkylation (BROD). In monkeys there were no sex-differences in untreated, phenobarbitone (PB)- or beta-naphthoflavone (BNF)-treated animals in AROD activities, or in individual P450 proteins detected by immunoblotting. Basal MROD and EROD activities varied by less than 7-fold between the five species, but the comparative pattern of basal MROD, EROD, PROD and BROD activities (the "MEPB profile") was very species-specific, with monkeys being similar to rats but different from man, mouse and hamster. The induction of AROD activities by PB and BNF was also highly species-specific. Monkeys expressed constitutive proteins immunorelated to the CYP1A, CYP2A, CYP2B, CYP2C and CYP3A sub-families (human CYP2A6 cross-reacted with the anti-rat CYP2B1 antibodies used, and so CYP2A and CYP2B forms could not be separately identified in the monkey). Single constitutive immunoblot bands were identified in monkey for CYP1A (54 kDa), CYP2A/CYP2B (51 kDa) and CYP3A (51 kDa), respectively, but two strong (51 and 52 kDa) plus two weak (49 and 49.5 kDa) bands were shown for CYP2C. Human liver expressed CYP1A2 (54 kDa), CYP2A6 (51 kDa), CYP3A4 (50.5 kDa) and three CYP2C9-immunorelated protein bands (48, 50 and 54 kDa). In monkeys BNF induced the 54 kDa CYP1A protein and CYP1A-dependent MROD, EROD and PROD activities (18-, 15- and 6-fold increases in activity, respectively), whereas PB strongly induced the 51 kDa CYP2A/CYP2B protein but did not induce PROD activity. PB also induced non-constitutive CYP2A/CYP2B protein bands at 49 and 52 kDa in some monkeys. BROD activity was induced less that four-fold by either PB or BNF in monkeys. In conclusion, cynomolgus monkeys expressed a range of constitutive CYP1A, CYP2A or CYP2B, CYP2C and CYP3A proteins similar to man, and a range of AROD monooxygenase reaction rates similar to both man and rat, but the basal MEPB profile of AROD activities in monkeys was more similar to rat than to man. MROD and EROD were good measures of CYP1A induction by polycyclic aromatic hydrocarbons in cynomolgus monkeys, but neither PROD nor BROD were indices of CYP2B induction by PB.

Hepatocytes isolated from both phenobarbital-induced and control rats were short-term cultured and exposed to cocaine (8–2000 μM) for varying times. Intracellular lactate dehydrogenase activity, free calcium levels ([Ca 2+ ] i ), reduced glutathione (GSH) and lipid peroxidation were investigated to evaluate the toxic effect of cocaine on hepatocytes. Cytochrome P450 induction by phenobarbital potentiated the in vitro cytotoxicity of cocaine by a factor of 13 (IC 50 = 84 μ M induced cells vs 1100 μM in non-induced cells). This difference in the susceptibility of the two types of hepatocytes to cocaine correlated well with the activity of cytochrome P450 2 B 1 2 . Rapid depletion of GSH, reaching 30% of the control levels, and massive lipid peroxidation thereafter were the two most remarkable phenomena preceding cell death in phenobarbital-induced hepatocytes. On the other hand, a sustained rise in [Ca 2+ ] i starting 2 hr after incubation with cocaine was the most noteworthy finding in non-induced liver cells. We suggest two different pathways for cocaine hepatotoxicity: in phenobarbital-induced hepatocytes oxidative metabolism of the drug causes GSH depletion, subsequent extensive lipid peroxidation and cell death, at low concentrations of cocaine. In non-induced hepatocytes these changes are less relevant, and the major alteration caused by cocaine is a non-transient rise in [Ca 2+ ] 1 that is evident at higher concentrations of the drug.

Rociverine [2-(diethylamino)-1-methylethyl cis-1-hydroxy [bicyclohexyl]-2-carboxylate] citrate (ROC) is an antispasmodic agent therapeutically active in humans at doses of 0.5-1 mg/kg. This study investigated the effect of acute administration of the drug on hepatic microsomal cytochrome P450 (P450)-catalysed drug metabolism. Only high doses (> or = 100 mg/kg) of ROC were able to induce in rats the hepatic microsomal pentoxyresorufin O-depenthylase (PROD) and 16 beta-testosterone hydroxylase activities both associated with P4502B1/2 and the erythromycin N-dimethylase (ErD) and 2 beta-testosterone hydroxylase activities both dependent on P4503A1/2. However, at 100 and 200 mg/kg of ROC, the 16 beta-testosterone hydroxylase and PROD were the most induced activities, suggesting that P4502B1/2 are the isoforms most sensitive to ROC induction. Accordingly, ROC treatment enhanced, in a dose-dependent manner, the amount of P4502B1/2 and 3A1/2 in microsomes as assayed by western blotting. The northern blot analysis of ROC-treated rat liver showed that the P4502B1/2 induction appears to be regulated at the mRNA level as in the induction by phenobarbital (PB). The oxidative metabolism of ROC with hepatic microsomes from control or PB- and ROC-induced rats resulted in a N-deethyl ROC derivative (major metabolite) and an unknown minor ROC derivative. The kinetic parameters for the N-deethylation of ROC were studied with purified P4502B1 and with microsomes from control or rats treated with various inducers (phenobarbital, ethanol, beta-naphthoflavone, dexamethasone and rociverine). It was found that phenobarbital-, dexamethasone- and rociverine-induced microsomes deethylated ROC with a Vmax about five times higher than that (0.9 nmol/min/mg protein) of control microsomes, although with a similar affinity (Km approximately 0.3 mM). In a reconstituted system, the purified P4502B1 metabolized ROC with a high deethylation rate (22 nmol/min/nmol P450). Moreover, the ROC deethylation was inhibited by compounds such as hexobarbital, metyrapone and triacetyloleandomicin, selective inhibitors for P4502B and/or P4503A enzymes. On the other hand ROC, when added in vitro, inhibited the 16 beta- and 2 beta-testosterone hydroxylases and the PROD and ErD activities. Taken together, these results indicate that the ROC-inducible P4502B and P4503A are involved in ROC deethylation. In conclusion, it has been demonstrated that ROC is a weak phenobarbital-like inducer of P450, probably able at high and reiterated doses to alter its own metabolism, at least in the rat liver.

Sex differences in the diabetes-induced changes in hepatic cytochrome P450 proteins were investigated in rats treated with streptozotocin. Changes in specific cytochrome P450 proteins were monitored using diagnostic substrates and immunologically utilizing specific polyclonal antibodies. When expressed in terms of nmoles of total cytochrome P450, ethoxyresorufin O-deethylase activity was increased by treatment with streptozotocin, the extent of induction being the same in the two sexes. In contrast, lauric acid hydroxylase and ethylmorphine N-demethylase activities were induced only in the male rat. Finally, p-nitrophenol hydroxylase and pentoxyresorufin O-dealkylase were enhanced by the same treatment in both sexes, the effect being more pronounced in the male. These findings indicate that sex-specific changes in certain cytochrome P450 proteins exist in response to insulin-dependent diabetes but these cannot, however, be ascribed to sex differences in the severity of diabetes induced by streptozotocin since the degrees of hyperketonaemia and hyperglycaemia were the same in the two sexes. These are likely to reflect sex-specific differences in growth hormone and triglyceride levels in the diabetic animals.

The inhibition of the P450 1A1 dependent de-ethylation of 7-ethoxyphenoxazone (7EPO) and the P450 2B1 dependent de-pentylation of 7-pentoxyphenoxazone (7PPO) by 1-ethynylnaphthalene (1EN), 2-ethynylnaphthalene (2EN), 1-ethynylanthracene (1EA), 2-ethynylanthracene (2EA), 9-ethynylanthracene (9EA), 2-ethynylphenathrene (2EPh), 3-ethynylphenanthrene (3EPh), 9-ethynylphenanthrene (9EPh), 1-ethynylpyrene (1EP) and 2-ethynylpyrene (2EP) was studied in hepatic microsomal preparations from rats. Although all of the polycyclic aromatic acetylenes studied inhibited the dealkylation of 7EPO or 7PPO, only some of the acetylenes produced a mechanism-based irreversible inactivation (suicide inhibition) of the P450 dependent dealkylation of 7EPO or 7PPO. Of the molecules tested, only 1EP, 1EN, 2EN, 2EPh and 3EPh were effective suicide inhibitors of the P450 1A1 dependent de-ethylation of 7EPO and only 1EN, 2EN, 1EA and 9EPh were effective suicide inhibitors of the P450 2B1 dependent de-pentylation of 7PPO. In addition to the size and shape of the polycyclic aromatic ring system, placement of the carbon--carbon triple bond on the ring system was critical for suicide inhibition. In contrast to 1EP, 2EP was not a mechanism-based inhibitor of P450 1A1; 9EPh, but not 2EPh or 3EPh, was a suicide inhibitor of P450 2B1. None of the aryl acetylenes tested produced heme destruction under assay conditions that produced the suicide inhibition of the P450 dependent 7EPO or 7PPO dealkylation activities. Because a precise orientation of the terminal acetylene is required to produce suicide inhibition without heme destruction, acetylenic suicide inhibitors can potentially be used to differentiate between P450 isozymes and to establish some distinguishing geometric features of the active site of these isozymes.

Exposure of cultured primary hepatocytes from Ah-responsive male C57BL/10ScSn mice to a polychlorinated biphenyl (PCB) mixture (Aroclor 1254) at 0.1-20 micrograms/mL for up to 96 hr induced cytochrome P4501AI-mediated activity (ethoxyresorufin O-deethylase, EROD) up to 50-fold. In contrast, pentoxyresorufin O-dealkylase (PROD), which in some circumstances is a measure of phenobarbitone-induced cytochrome P450 isoenzymes, was induced only 5-fold. There were similar findings on EROD activities with the pure compounds 3,3',4,4',5,5'-hexachlorobiphenyl, 3,3',4,4',5,5'-hexabromobiphenyl and 3,3',4,4'-tetrachlorobiphenyl(TCB) and also beta-naphthoflavone but not with 2,2',4,4'-TCB or phenobarbitone. The higher concentrations of Aroclor 1254 were also associated with cytotoxicity as estimated by release of alanine aminotransferase (ALT) into the medium. Unlike in C57BL/10ScSn hepatocytes induction of EROD and cytotoxicity was minimal in hepatocytes from the Ah-non-responsive strain DBA/2. Although in vivo the hepatic toxicity and carcinogenicity of polyhalogenated aromatics are markedly potentiated by iron, no enhancement of the cytotoxicity of Aroclor 1254 towards C57BL/10ScSn hepatocytes by iron was observed in vitro. However, iron caused decreased EROD activities and possibly cytochrome P4501AI (as judged by Western blotting) as in vivo. Even in the presence of iron and the haem precursor 5-aminolaevulinic acid (5-ALA) there was no development of uroporphyria in this system although this occurs with Aroclor in vivo and is enhanced by iron. Accumulation of uroporphyrin did occur after extended culture of C57BL/10ScSn hepatocytes on matrigel for 8 days in the presence of 5-ALA and Aroclor 1254 but again no potentiation by iron was observed. Thus, although culture of Ah-responsive and -non-responsive hepatocytes mimics some aspects of the mechanisms of in vivo toxicity of PCBs, there is some unknown associated influence of iron metabolism which cannot, as yet, be produced in vitro but which is of importance in vivo.

The non-sedating anti-histamine, loratadine [ethyl 4-(8-chloro-5,6-dihydro-11H-benzo[5,6]-cyclohepta[1,2-b]pyridin- 11-ylidene-1-piperidinecarboxylate], was administered orally in the diet to mature male rats at dosages of 4, 10 and 25 mg/kg/day for 2 weeks. The effects of these treatments on liver microsomal cytochrome P450 were evaluated by immunochemical and biochemical techniques, and were compared with the effects of treating rats with three different inducers of cytochrome P450, namely phenobarbital, 3-methylcholanthrene and dexamethasone. Treatment of rats with loratadine caused a dose-dependent increase in the levels of P450 2B1 and 2B2, the major phenobarbital-inducible P450 enzymes, as determined by Western immunoblotting. At the highest dosage tested, loratadine was less effective than phenobarbital as an inducer of 2B1 and 2B2, although the induction of these proteins could be detected immunochemically even at the lowest dosage of loratadine tested. Consistent with these observations, treatment of rats with loratadine caused a dose-dependent increase in the rate of two reactions that are catalyzed predominantly by 2B1/2, namely testosterone 16 beta-hydroxylation and 7-pentoxyresorufin O-dealkylation. At the highest dosage tested, loratadine caused a 7.3- and 8.5-fold increase in the rate of testosterone 16 beta-hydroxylation and 7-pentoxyresorufin O-dealkylation, respectively, compared with a 22- and 45-fold increase caused by phenobarbital treatment. Treatment of rats with loratadine caused a 1.4- to 2.0-fold increase in the 2 beta-, 6 beta- and 15 beta-hydroxylation of testosterone, which was associated with a similar increase in the levels of immunoreactive P450 3A1 and/or 3A2. As an inducer of P450 3A1/2, loratadine was slightly less effective than phenobarbital, and was considerably less effective than dexamethasone, which caused a 10- to 33-fold increase in testosterone 2 beta-, 6 beta- and 15 beta-hydroxylase activity. At the dosages tested, loratadine did not increase the levels of P450 1A1, the major 3-methylcholanthrene-inducible P450 enzyme, as determined by Western immunoblotting. The rate of 7-ethoxyresorufin O-dealkylation, which is catalyzed predominantly by P450 1A1, increased 1.9-fold after loratidine treatment, but this increase was less than that caused by phenobarbital treatment (2.2-fold), and was considerably less than that caused by 3-methylcholanthrene treatment (33-fold). The effects of treating mature male mice with loratadine on liver microsomal cytochrome P450 resembled the effects observed in rats. These results indicate that loratadine is a phenobarbital-type inducer of liver microsomal cytochrome P450 in rats and mice.

Human embryonic tissues were investigated during the period of organogenesis with a combination of substrate probes, selective inhibitors and immunoprobes in terms of their capacity to express functional P450 isoforms. A series of phenoxazone ethers utilized as substrate probes revealed that human embryonic hepatic, pulmonary, renal, adrenal and cardiac tissues each contained a complement of functional P450 isoforms when analyzed between days 50 and 60 of gestation. Preparations of each of these tissues contained isoforms capable of catalyzing O-demethylation, O-deethylation, O-depentylation and O-debenzylation of the respective phenoxazone ethers. Investigations with chemical inhibitors and inhibitory antibodies as well as comparisons with vector-expressed, human P450 isoforms suggested that isoforms of P450 subfamilies 1A, 2B, 2C or 3A were not major contributors to any of the observed reactions. The P450-dependent reactions studied exhibited several unexpected and unusual characteristics including a preference for NADH over NADPH as the initial electron donor. Results were consistent with the concept that conceptal-specific P450 isoforms participate in the human embryonic O-dealkylation/debenzylation probe reactions investigated.

Our previous studies have shown that drug-metabolizing activities in the eye are highest in the ciliary body, a tissue responsible for aqueous humor production. In this work, we have separated nonpigmented epithelial (NPE) cells and pigmented epithelial (PE) cells from porcine ciliary body and determined basal and induced activities of 7-ethoxyresorufin (ER) O -dealkylase, 7-pentoxyresorufin (PR) O -dealkylase, and UDP-glucuronosyl transferase (UDP-GT) using primary cultures of separated cells. ER and PR activities were associated primarily with NPE cells and were very low in PE cells. Treatment of NPE cells with phenobarbital (PB) for 48 hr resulted in about a 4-fold increase PR O -dealkylase activity but only a 1.3-fold rise in ER O -dealkylase activity. Conversely, 3-methylcholanthrene (MC) treatment augmented the ER O -dealkylase activity of NPE cells 6 times over the basal activity in 48 hr but had little effect on PR O -dealkylase activity. Both NPE and PE cells had low basal UDP-GT activities. UDP-GT activity increased about 5-fold in PB-treated PE cells and about 4-fold in PB-treated NPE cells in 48 hr. The results of MC treatment were similar to those of PB treatment; enhancement of UDP-GT was more pronounced in PE cells than in NPE cells. Induction by PB and MC of ER O -dealkylase, PR O -dealkylase and UDP-GT activities in ciliary NPE and PE cells was inhibited almost completely by 3.5 μM cyclohexamide and 40 nM actinomycin D. The heterogeneous distribution of these enzymes suggests that a harmonius interplay between NPE and PE cells is important for metabolic detoxification of blood plasma prior to aqueous humor formation.

Changes in the isoenzyme pattern of cytochrome P450 during culture were investigated in primary cultures of rat hepatocytes, measuring specific enzyme activities in microsomes prepared from cultured cells as well as in intact monolayers. Assays of 7-ethoxyresorufin O-deethylation (EROD), 7-pentoxyresorufin O-depentylation (PROD), aniline 4-hydroxylation (AH) and the specific regioselective hydroxylation of testosterone were used as representatives of the activities of seven isoenzymes of cytochrome P450. The isoenzyme profile expressed as catalytic activities was qualitatively and quantitatively similar in microsomes obtained from freshly isolated hepatocytes in comparison with microsomes obtained from whole livers of untreated rats. There was a relatively high activity in EROD, AH and the oxidation of testosterone at the 7 alpha, 2 alpha, 6 beta, 16 alpha and 17 sites (androstenedione). During culture, these microsomal enzyme activities declined at a similar rate to ca. 50% of the activities of microsomes prepared from freshly isolated hepatocytes after 24 hr and to 15% after 96 hr. The overall decline of cytochrome P450-dependent activities during culture was not accompanied with gross changes in catalytic profile. Determining the same drug-metabolizing activities directly in intact hepatocyte monolayers revealed a much higher metabolic rate for all measured P450-dependent activities. The profile of the catalytic activities was essentially the same as measured in microsomes prepared from cultured hepatocytes. The relatively low activity towards the 7 alpha site of testosterone measured in intact hepatocytes, however, remained constant during culture. Determination of enzyme activities directly in intact hepatocytes is a convenient way of studying changes in monooxygenase activities of different P450 isoenzymes in vitro.

In cultured human hepatoma cells phenolphthalein glucuronidation was increased 3-fold by 2 mM phenobarbitone (PB) in the culture medium but not by 25 μM benz( a )anthracene (BA), while 1-naphthol glucuronidation was not increased by either PB or BA. Ethoxyresorufin O -deethylation (EROD) was increased 15-fold by BA but not by PB, while the O -dealkylations of pentoxyresorufin (PROD) and benzyloxyresorufin (BROD) were increased by either PB or BA. The BROD activity increased by BA was sensitive to inhibition by α-naphthoflavone whereas that induced by PB was not. This suggests induction of different cytochrome P-450 isoenzymes. Control Hep G2 cells had similar glucuronide conjugation and cytochrome reductase activities to freshly isolated human adult hepatocytes, but had lower O -dealkylation and elevated musomal epoxide hydrolase activities.

The imidazole N-substituted antifungal agents ketoconazole, miconazole and clotrimazole have been shown to be potent inhibitors of oxidative metabolism by both a phenobarbital-induced cytochrome P-450 (P-450b) and a 3-methylcholanthrene-induced cytochrome P-448-protein (P-450c) in reconstituted systems. All three compounds inhibited the cytochrome P-450b-dependent 7-pentoxyresorufin-O-dealkylase and the cytochrome P-450c-dependent 7-ethoxyresorufin-O-deethylase activities. When 7-benzyloxyresorufin and 7-ethoxycoumarin were employed as substrates with both cytochrome preparations, all three antifungal compounds exhibited selective inhibition of the cytochrome P-450b preparation; ketoconazole was always the weakest inhibitor. The three antifungal agents were also shown to elicit a type II difference spectral interaction with both isoenzymes, the magnitude of the spectral interaction being greater with the cytochrome P-450b preparation.

A chemically defined medium developed for the maintenance of differentiated adult rat hepatocytes (T1) was compared with two commercially available media (Waymouth 752/1 and Leibovitz L-15) for maintenance of cytochrome P-450 metabolic activity in cultured hepatocytes. Specific metabolic activities of initially isolated cells and 72-hr control and phenobarbital-treated cultures were determined with 7-ethoxycoumarin, 7-ethoxyresorufin, and 7-pentoxyresorufin as substrates. Control and phenobarbital-treated cultures in T1 medium had a higher metabolic activity towards each of the three substrates than comparable cultures in the other media. These studies indicated that the metabolic activity and the response to phenobarbital of the major isozyme of the phenobarbital-inducible family of cytochrome P-450 were maintained in hepatocytes in T1 medium. However, there was anomalous expression and induction by phenobarbital of the major 3-methylcholanthrene-inducible isozyme, cytochrome P-450c, in cultured hepatocytes in each of the three media tested, but this response was more pronounced in T1 medium. In conclusion, the regulation of cytochrome P-450 metabolic activity in cultured hepatocytes was shown to be dependent on the composition of the culture medium.